Apparatus and sensing devices for measuring fluorescence lifetimes of fluorescence sensors

ABSTRACT

An apparatus and devices for measuring fluorescence lifetimes of fluorescence sensors for one or more analytes, the apparatus comprising (c) one or more reference systems ( 3,6,7 ), said reference systems each comprising one or more reference light sources ( 3 ) and being adapted to receive one or more excitation signals ( 1   a ), to produce reference optical signals ( 6   b ) in response thereto, and to produce one or more electrical reference output signals ( 7   b ) in response to one or more excitation signals ( 1   a ); and (d) or more phase detectors ( 10 ), said phase detections being adapted to detect one or more delays of said one or more electrical output signals of said one or more fluorescence sensor systems and said one or more reference systems, and to produce one or more phase output signals; and a method of measuring concentration of one or more analytes using such apparatus and/or devices.

BACKGROUND OF THE INVENTION

The present invention relates to an apparatus for measuring fluorescencelifetimes of fluorescence sensors for one or more analytes, fluorescencelifetime sensing devices for sensing fluorescence light of fluorescencesensors for one or more analytes, and a method of measuringconcentration of said one or more analytes using such apparatus and/ordevices.

In the present context, the terms analyte or analytes are intended todesignate analytes in a broad sense including chemical substances suchas biomolecules, blood gases e.g. O₂, CO₂; pH, salt ions e.g Na⁺ andCl⁻; and physical parameters such as temperature and pressure.

THE TECHNICAL FIELD

In biological and industrial processes, efficient control of theprocesses requires simultaneous monitoring of several key-parametersthereof, e.g. parameters such as temperature, pH, PO₂ and CO₂. Numeroussensor systems have been developed for such monitoring systems. However,they are usually based on commercially available discrete sensors, eachof which is capable of sensing a single key-parameter. Consequently,there is a need for multi-analyte sensors capable of sensing severalkey-parameters simultaneously.

In particular in modern biotechnology relating to e.g. tissueengineering and genetic technology, cultivation is often performed inparallel systems with numerous batches being processed simultaneously.For efficient control of processes in such systems, a large number ofanalyses are required. Normally, such parallel systems require largeprocess volumes. However, there is a trend to scale down process volumesto smaller volumes, e.g. less than 1 liter, to obtain more optimalprocess economy. For such smaller process volumes, however, even smallersample volumes are required, often sample volumes of only fewmilliliters.

In addition, cultivation of tissues, cells, bacteria or othermicroorganisms in laboratories often uses small volume systems of lessthan 100 ml volume. Consequently, the sensors are required to besuitably small for use with such small sample volume.

Consequently, the use of conventional sensors is limited by bothavailable physical space, small sample volumes and especially forparallel systems, the multiplication of sensors increasing costs. Thesefactors combined have made precise process control unavailable for manyapplications.

Multi-analyte sensors based on measurement of fluorescence intensitymeasurements are known but they suffer from being influenced by drift ofthe intensity of the excitation source, photo-bleaching of activecomponent of the sensor, and drift of detector efficiency.

Other multi-analyte sensors are based on fluorescence lifetime. Thesesensors solve the problem of sensitivity to drift in the opto-electroniccomponents whereby the need for frequent re-calibrations is reduced.

In fluorescence lifetime based sensors, the chemical analytes aredetected by measuring a change in the fluorescence lifetime of theutilized fluorophores. Fluorescent lifetime measurements are superior tointensity measurements in a number of aspects. Firstly, intensity driftin the excitation source and drift in the efficiency of the detectors donot affect the measurements. Secondly, photo-bleaching of thefluorophores only limits the total operational lifetime of the sensor(not the fluorescence lifetime of the fluorophore itself), and not themeasurements. The sensor is operational as long as the sensor chemistryis capable of delivering a signal sufficiently high compared to thenoise in the system. The need for re-calibration due to photo-bleachingis therefore reduced which is very important in e.g. industrial processmonitoring applications where sensor systems are to be operatedcontinuously for long periods of time.

There are mainly two ways of performing time-resolved fluorescencemeasurements; time-domain and frequency-domain measurements (see e.g.Lakowicz, Joseph R., Principles of fluorescence spectroscopy, SecondEdition, Kluwer Academic/Plenum Publishers, New York 1999). Presently,the frequency-domain technique provides a robust and inexpensivetechnique with less stringent component demands compared to thetime-domain technique.

When utilising frequency-domain fluorescence lifetime measurements wherephase resolutions in the order of fractions of a degree is needed, thephase of the excitation source has to be known. Normally this isobtained by utilising phase stable excitation sources (e.g. largelasers) but this solution is not a suitable solution for a mass-producedindustrial product. In such systems, light emitting diodes (LEDs) aremore suitable, as they are compact, cheap and efficient.

They do, however, suffer from a bias current dependent phase, makingprecise lifetime measurements with LEDs very difficult.

PRIOR ART DISCLOSURES

EP 0 448 923 discloses a method, sensor and apparatus for detectingbiological activities of a specimen by introducing a sample of thespecimen into a sealed, transparent container containing a culturemedium enabling metabolic processes in presence of microorganisms in thesample. Changes are monitored over time of concentrations of substancesinvolved in the metabolic processes, e.g. O₂, CO₂ and pH. A change ismeasured in fluorescence intensity of at least one activable, inertfluorophore and at least one indicator component that changes theiroptical characteristics in response to changes in concentration of atleast one such substance in the container. As these systems are bases onmeasurement of changes in fluorescence intensity, they are sensitive todrift in the intensity of the excitation source, photo-bleaching of thefluorophore and drift in detector efficiency.

WO 99/06821 discloses a method and apparatus for fluorometricdetermination of a biological, chemical or physical parameter of asample comprising measuring the time or phase behaviour of at least twoluminescent materials having different decay times wherein at least theluminescent intensity of one of the luminescent materials (the sensorluminophore) responds to the parameter to be determined and at least theluminescent intensity and decay times of the others of the luminescentmaterials (the internal reference luminophore) usually those havinglonger decay times does not respond to the parameter to be determined.The intensity of the luminescence of the internal reference luminophorefunctions as an internal reference for the intensity of the luminescencefrom the sensor luminophore whereby a second light source or a secondlight detector can be avoided.

DISCLOSURE OF THE INVENTION Object of the Invention

It is an object of the present invention to seek to provide an improvedmethod and apparatus for measuring concentration of one or moreanalytes.

It is another object of the present invention to seek to provide animproved apparatus for measuring fluorescence lifetime of fluorescencesensors for one or more analytes for which influences of drift of theintensity of the excitation source, photo-bleaching of active componentof the sensor, and drift of detector efficiency are reduced.

It is still another object of the present invention to seek to providesuch an improved apparatus for measuring fluorescence lifetimes offluorescence sensors for one or more analytes simultaneously.

Further objects appear from the description elsewhere.

Solution According to the Invention

According to the present invention, these objects are fulfilled byproviding an apparatus for measuring fluorescence lifetimes offluorescence sensors for one or more analytes as defined in claim 1, theapparatus comprising

-   (a) one or more excitation light sources, said light sources being    adapted to produce one or more excitation signals, and optionally    further comprising beam adapting optics;-   (b) two or more fluorescence sensor systems, said sensor systems    each comprising one or more fluorescence sensors for sensing the one    or more analytes and being adapted to receive said one or more    excitation signals, to produce one or more optical sensor signals in    response thereto, and to produce one or more electrical output    signals in response to said optical sensor signals, said one or more    electrical output signals being delayed with respect to said one or    more excitation signals and being characteristics of the    fluorescence lifetimes of the one or more fluorescence sensors;-   (c) one or more reference systems, said reference systems each    comprising one or more reference light sources and being adapted to    produce one or more electrical reference output signals in response    to said one or more excitation signals to receive said one or more    excitation signals, to produce reference optical signals in response    thereto, and to produce one or more electrical reference output    signals in response to said one or more optical reference signals;    and-   (d) one or more phase detectors, said phase detectors being adapted    to detect one or more delays of said one or more electrical output    signals of said one or more fluorescence sensor systems and said one    or more reference systems, and to produce one or more phase output    signals.

It has turned out that by providing one or more reference systems, saidreference systems each comprising one or more passive reference lightsources and being adapted to produce one or more electrical referenceoutput signals in response to said one or more excitation signals; andby providing one or more phase detectors, said phase detectors beingadapted to detect one or more delays of said one or more electricaloutput signals of said one or more fluorescence sensor systems and saidone or more reference systems, it is obtained that the influences ofdrift of the intensity of the excitation source, photo-bleaching ofactive component of the sensor, and drift in the detector efficiency onthe phase detector outputs on said one or more phase output signals arereduced whereby an improved apparatus for measuring concentration of oneor more analytes is obtained.

Conversion of said one or more phase output signals to concentrationmeasures are known in the art, see e.g. Lakowicz, Joseph R., Principlesof fluorescence spectroscopy, Second Edition, Kluwer Academic/PlenumPublishers, New York 1999.

In general, conversion factors are based on calibration of phase outputsignals produced by samples containing known analytes including knownconcentrations of the analytes of interest. Also, conversion factors canbe based on absolute calibration using physical and chemical parametersof the sample and analytes.

Conversion factors are typically stored and retrieved forphase-to-concentration conversion by means known in the art, e.g. bycomputer and electronic storage media.

Preferred embodiments are defined in the sub claims.

Reference Systems

According to the present invention, the one or more reference systemseach comprise one or more passive reference light sources and areadapted to produce one or more electrical reference output signals inresponse to said one or more excitation signals.

Generally, the reference light source can be any suitable referencelight source which varies in a similar manner as the fluorescencesensors do with respect to all influences thereon except that of the oneor more analytes.

Reference light sources can be passive or active. Passive light sourcesare preferred as they usually involve fewer sources of variability.

In a preferred embodiment, said one or more reference systems comprise afluorophore, a phosphore, or both whereby reference systems having verysimilar behaviour as the sensor systems can be designed.

For examples, for reference systems wherein the passive reference lightsource comprises a fluorophore, e.g. fluorescein derivatives, rhodaminderivatives, or both, that are encapsulated in e.g. nano- ormicroparticles for shielding from influence of the environment, theresponse of the reference light source can be made similar to that ofthe fluorescence system for one or more analytes. As the fluorophoreshifts the reference wavelength, this solution also reduces straylightat the excitation wave-length.

In another preferred embodiment, said one or more reference systemcomprise one or more reflectors, said reflectors reflecting said lightof said one or more excitation light sources. This embodiment provides aparticularly simple reference light source for many applications.Further, this reference light source does not depend on the effect ofthe surroundings of the analyte on the fluorescence sensors.

The reflectors of the reference system can be of any suitable form thatprovides reflection. In still another preferred embodiment, said one ormore reflectors comprise of a diffuse reflector, a retro-reflector, orboth, whereby a reference system highly insensitive to changes in thespatial radiation characteristics of the excitation source is obtained.

In a particularly preferred embodiment, said one or more reflectorscomprise a mirror whereby a high level of optical control of thereference light is obtained, minimising the influence of straylight.

In still a further preferred embodiment, said one or more referencesystems are placed close to said one or more sensor systems whereby mostequal conditions are met for both the fluorescence sensors and thereferences, and the accuracy of the referencing is further improved.

Phase Detectors

According to the present invention, there is provided one or more phasedetectors, said phase detectors being adapted to detect one or moredelays of said one or more electrical output signals of said one or morefluorescence sensor systems and said one or more reference systems, andto produce one or more phase output signals.

Phase detectors are known in the art, see e.g. Stanford Research SystemsCatalogue 1998, Application Note #3, “About Lock-in Amplifiers” pp.193-204, the content of which is hereby incorporated by reference.

One or More Excitation Light Sources

The one and more excitation light sources comprise light source which isable to excite fluorophores and/or phosphores of the sensor systems.

An important parameter of the excitation light is its wavelength. Insome applications the fluorescence sensor can be designed, so that allsensor systems can utilize the same excitation light source, inparticular in case of multiple wavelength of the same light source.

In a preferred embodiment, the apparatus comprises one or more singleexcitation light sources for said sensor systems and reference systems.

In some applications, the single excitation light source might notcomprise the required multiple wavelengths which can excite the desiredfluorophores and phosphores. Consequently, more than one excitationsource is required.

In another preferred embodiment, the apparatus comprises one excitationlight source for each sensor system and each reference system wherebyoptimum excitation efficiencies can be obtained for each fluorescencesensor.

The one or more excitation light sources can be operated in time domain,frequency domain or both.

In a preferred embodiment, said one and more light sources comprise atleast one excitation light source adapted to operate in frequencydomain.

Fluorescence Sensors-Sensor Chemistry

The fluorescence sensor can be in any suitable form wherein the analytescan be sensed by the fluorescence sensor chemistry and provide acharacteristic emission of fluorescence light of the fluorophore inresponse of the presence of an analyte and the excitation light.

In a preferred embodiment, the one or more fluorescence sensors forsensing the one or more analytes are incorporated in an exchangeablesensor cap whereby sensors can be designed for the same system ofexcitation light sources, reference system, and phase detectors.

Generally, in the present context it is intended that the termfluorophore designates both fluorophore and phosphore, respectively.

Consequently, in a preferred embodiment, the one or more sensors forsensing the one or more analytes comprise a fluorophore, a phosphore, orboth.

Fluorescence sensor chemistry is known in the art, see e.g. WolfbeisOtto S. et. al., “Set of luminescence decay time based chemical sensorsfor clinical applications”, Sensors and Actuators B, Vol. 51, 1998, p.17-24

Fluorescence Sensor Systems

The fluorescence sensor systems can be designed in any suitable way thatallows one or more fluorescence sensors to sense one or more analytes,when brought into contact therewith, and to receive one or moreexcitation signals to produce one or more electrical output signals inresponse thereto. The one or more electrical output signals are delayedwith respect to said one or more excitation signals and they arecharacteristic of the fluorescence lifetimes of the one or morefluorescence sensors.

Preferred embodiments of the fluorescence sensor systems depend on theapplication.

In a preferred embodiment, the one or more fluorescence sensors forsensing the one or more analytes are incorporated in an exchangeablesensor cap whereby a sensor system which is easy to exchange isprovided. This is advantageous for easy and fast exchange of sensorchemistry when damaged, or if the configuration of the sensor is to bealtered for sensing of different analytes.

Particularly, said one or more fluorescence sensors for sensing the oneor more analytes comprise a fluorophores, a phosphore, or both.

In another preferred embodiment, said one or more sensor systemscomprise one or more sensors, one or more detectors, and one or morewaveguides between said one or more sensors and detectors whereby aparticular compact, robust multi-analyte sensor can be provided.

In still another embodiment, said one or more sensor systems compriseone or more light directing means, said light directing means directingsaid one or more excitation light signals to said one or more sensors.

In still another embodiment, said one or more light directing meansconsist of one or more reflective cones whereby a particular simplemeans of directing excitation light to the fluorescence sensors isprovided.

In still another embodiment, said one or more light directing meansconsist of one or more diffractive optical elements whereby a particularcompact apparatus can be provided.

Application Systems

The apparatus according to the present invention can be implemented inany suitable way that allows said fluorescence sensor systems andreference systems to become implemented.

The present invention can be utilized within a large variety ofapplication areas, including but not limited to, wastewater cleaningplants, drinking water processing, industrial fermentation tanks,general food processing, modified atmosphere packed food (MAP),micro-reactor scanning systems, tissue engineering, etc.

In a preferred embodiment, said one or more sensor systems and saidreference system are incorporated in a flow cell whereby a fluid or gasflowing continuously can be monitored for multiple analytes.

In another preferred embodiment, said one or more sensor systems andsaid reference system are incorporated in a micro bioreactor wherebypreviously unavailable on-line monitoring of multiple analytes becomespossible.

In still another preferred embodiment, said one or more sensor systemsand said reference system are incorporated in a micro fluid-channelsystem.

In still another preferred embodiment, said one or more sensors of thesensor systems are wholly or partially covered with one or moresemi-permeable membranes. Such membranes can be silicone or Teflonmembranes for measuring gaseous or other neutral species and forimproving the selectivity (gas permeable but with ion shieldingproperties). In addition, such membranes can include blackanalyte-permeable layers to improve photostability to shield the sensorfrom intrinsic sample fluorescence, and to avoid a too high level ofambient light

Time-Resolved Fluorescence Measurement

There is basically two ways of performing time-resolved fluorescencemeasurements: time-domain and frequency-domain measurements (see e.g.Lakowicz, Joseph R., Principles of fluorescence spectroscopy, SecondEdition, Kluwer Academic/Plenum Publishers, New York 1999).

Time-resolved fluorescence measurements utilise a short pulse forexcitation of the fluorophore and the decay of the fluorescence lightfrom the fluorophor is then measured. This technique requires a lightsource capable of emitting very short pulses of light and very fastsampling detection electronics.

Frequency-domain measurements utilize a continuously modulated lightsource for exciting the fluorophore. The fluorescence emitted from thefluorophore will then be delayed compared to the excitation light due tothe fluorescence lifetime of the fluorophor. This delay can be measuredas a phase shift between the excitation light and the emittedfluorescence. This technique is advantageous for mass produced sensorscompared to the time-domain technique, as both the excitation lightsource and the detection electronics are significantly cheaper.

Fluorescence Lifetime Sensing Device Comprising an Optical LightBeam-Adapting System which Comprises a Reflective Surface

In still another aspect according to the present invention, theseobjects are fulfilled by providing a fluorescence lifetime sensingdevice for sensing fluorescence lifetimes of fluorescence sensors forone or more analytes, the sensor device comprising:

-   a fluorescence sensor system comprising one or more fluorescence    sensors, said sensors being adapted to sense the one or more    analytes and produce fluorescence light in response thereto;-   a phase reference system comprising a passive reference light    source;-   an optical light beam-adapting system providing excitation lights    for the fluorescence sensors and reference light for said phase    reference system;-   a detection system comprising detectors for detecting said    fluorescence light from said fluorescence sensors and reference    light from said phase reference system; and-   an optical sensor and reference signal guiding system, said guiding    system guiding said fluorescence light and said reference light to    said detectors;-   wherein said optical light beam-adapting system comprises a    reflective surface directing said excitation light to the    fluorescence sensors and said reference light to said phase    reference system.

It turns out that when said optical light-beam adapting system comprisesa reflective surface directing said excitation light to the fluorescencesensors and said reference light to said phase reference system, aparticular simple and robust sensing device can be provided.

The reflective surface can be any reflective surface that is able todirect said excitation light to said fluorescence sensors and saidreference light to said phase reference system.

In a preferred embodiment, said reflective surface comprises the outersurface of a cone whereby a particularly simplified multi-analyte sensorcan be provided.

In another preferred embodiment, the optical light beam-adapting systemcomprises optical fibres whereby said excitation light and referencelight can easily be guided to the fluorescence sensor and phasereference system, respectively. Furthermore, utilization of opticalfibres for transport of excitation light enables remote location of theexcitation source in relation to the detectors, reducing excitationsource induced electromagnetic noise in the detection circuits.

Similarly, in another preferred embodiment, said optical sensor andreference signal guiding system comprises optical fibres, whereby aparticular effective lightguiding system is obtained, reducing thephoto-bleaching of the sensor chemicals as they can then providesufficient signal with less excitation light.

In a preferred embodiment, this sensing device comprises one or morephase detectors as defined for the apparatus according to the presentinvention; said phase detectors being adapted to detect one or moredelays of said one or more electrical output signals of said one or morefluorescence sensor systems and said one or more reference systems, andto produce one or more phase output signals.

Fluorescence Lifetime Sensing Device Comprising One or More FluorescenceSensors Incorporated in an Exchangeable Cap

In still another aspect according to the present invention, theseobjects are fulfilled by providing a fluorescence lifetime sensingdevice for sensing fluorescence lifetimes of fluorescence sensors forone or more analytes, the sensor device comprising

-   a fluorescence sensor system comprising one or more fluorescence    sensors, said sensors being adapted to sense the one or more    analytes and produce fluorescence light in response thereto;-   a phase reference system comprising a passive reference light    source;-   an optical light beam-adapting system providing excitation lights    for the fluorescence sensors and reference light for said phase    reference system;-   a detection system comprising detectors for detecting said    fluorescence light from said fluorescence sensors and reference    light from said phase reference system; and-   an optical sensor and reference signal guiding system, said guiding    system guiding said fluorescence light and said reference light to    said detectors;-   wherein said one or more fluorescence sensors are incorporated in an    exchangeable cap.

It turns out that when said one or more fluorescence sensors areincorporated in an exchangeable cap, a particular flexible sensingdevice with low operating costs can be provided.

In a particularly preferred embodiment, said reference light source isincorporated in said exchangeable cap whereby said fluorescence sensorsand said reference light source can be brought closely together andprovide an improved referencing with approximately similar conditionsfor the fluorescence sensor and the reference.

In a preferred embodiment, this sensing device comprises one or morephase detectors as defined for the apparatus according to the presentinvention; said phase detectors being adapted to detect one or moredelays of said one or more electrical output signals of said one or morefluorescence sensor systems and said one or more reference systems, andto produce one or more phase output signals.

Fluorencence Lifetime Sensing Device Comprising an Optical LightBeam-Adapting System and an Optical Sensor and Reference Signal GuidingSystem One of Which, or Both, are Incorporated in an Diffractive OpticalElement

In still another aspect according to the present invention, theseobjects are fulfilled by providing a fluorescence lifetime sensingdevice for sensing fluorescence lifetimes of fluorescence sensors forone or more analytes, the sensor device comprising

-   a fluorescence sensor system comprising one or more fluorescence    sensors, said sensors being adapted to sense the one or more    analytes and produce fluorescence light in response thereto;-   a phase reference system comprising a reference light source;-   an optical light beam-adapting system providing excitation lights    for the fluorescence sensors and reference light for said phase    reference system;-   a detection system comprising detectors for detecting said    fluorescence light from said fluorescence sensors and reference    light from said phase reference system; and-   an optical sensor and reference signal guiding system, said guiding    system guiding said fluorescence light and said reference light to    said detectors;-   wherein said optical light beam-adapting system, said optical sensor    and reference signal guiding system, or both, are incorporated in an    diffractive optical element.

It turns out that when said optical light beam-adapting system, saidoptical sensor and reference signal guiding system, or both, areincorporated in a diffractive optical element, a particular compactsensing device can be provided. Furthermore, this design reduces thenumber of components which lower production cost and increasereliability.

Diffractive optical elements can be produced by methods known in theart, see e.g. Babin, S. V. “Data preparation and fabrication of DOEusing electron-beam lithography”, Optics and Lasers in Engineering, Vol.29 Issue 4-5, 1998, pp. 307-324, and Taghizadeh, M. R. et al. “Designand fabrication of diffractive optical elements”, MicroelectronicEngineering, Vol. 34, Issue 3-4, 1997, pp. 219-242.

In a preferred embodiment, said optical light beam-adapting systemcomprises a stacked planar integrated optical layer structure which isespecially advantageous for mass production.

Stacked planar integrated optical layer structures can be produced bymethods known in the art, see e.g. Sinzinger, S. J. J “Microoptics”,Wiley-VCH, 1999.

In a particularly preferred embodiment, said layer structure comprisesan electronic layer, a detector layer, a light source, a diffractiveoptical element, a sensor and reference layer and a filter layer.

In a preferred embodiment, this sensing device comprises one or morephase detectors as defined for the apparatus according to the presentinvention; said phase detectors being adapted to detect one or moredelays of said one or more electrical output signals of said one or morefluorescence sensor systems and said one or more reference systems, andto produce one or more phase output signals.

Method of Measuring Concentration

In another aspect of the present invention there is provided a method ofmeasuring the concentration of one or more analytes, the methodcomprising

-   (a) providing an apparatus as defined according to the invention, or    a device according to the invention;-   (b) applying said one or more excitation light signals to said one    or more fluorescence sensor systems and to said one or more passive    reference light sources;-   (c) applying said one or more electrical output signals of said one    or more fluorescence sensor systems and said one or more reference    systems to said one or more phase detectors;-   (d) determining said one or more delays by said one or more phase    output signals; and-   (f) comparing said one or more determined delays with delay    calibration data of known concentrations of the one or more    analytes,-   whereby particular accurate concentration measurements can be    obtained.

Fluorescence Lifetime Measurement

Measurements of fluorescence lifetime are known in the art. They includemeasurements in the time-domain and/or frequency domain, see e.g.Lakowicz, Joseph R., Principles of fluorescence spectroscopy, SecondEdition, Kluwer Academic/Plenum Publishers, New York 1999.

In a preferred embodiment, the present invention is based on frequencydomain measurement which allows a robust and inexpensive technique to beimplemented with less stringent component demands compared to thetime-domain technique.

In particular, phase referencing according to the present inventionallows very accurate determination of the phase of the excitation sourcewhereby phase resolutions in the order of fractions of a degree can beobtained.

Consequently, fluorescent lifetime measurements based on frequencydomain and referencing according to the present invention provide anumber of advantages.

Firstly, intensity drift in the excitation source and drift in theefficiency of the detectors do not affect the measurements. Secondly,photo-bleaching of the fluorophores only limits the total lifetime ofthe sensor, and not the measurements. The sensor is operational as longas there is provided a sufficiently high signal compared to the noise inthe system. The need for re-calibration due to photo-bleaching istherefore strongly reduced, which is very important in e.g. industrialprocess monitoring applications where sensor systems are to be operatedcontinuously for long periods of time.

Fluorescence lifetime measurements of multi-analyte sensors may becombined with other techniques for sensing analytes such as techniquesbased on intensity, polarisation, optical rotation, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following, by way of examples only, the invention is furtherdisclosed with detailed description of preferred embodiments. Referenceis made to the drawings in which

FIG. 1 shows a flow chart diagram of information flow in a multi-analytesensor device according to an embodiment of the present invention;

FIG. 2 illustrates delay between an electrical sensor output signal anda phase reference signal infrequency domain fluorescence lifetimemeasurement;

FIG. 3 illustrates a preferred embodiment of a fluorescence sensor andreference system in a longitudinal cross sectional view;

FIGS. 4A-4C illustrate a preferred embodiment of an exchangeable sensorcap illustrated in FIG. 3;

FIGS. 5A and 5B illustrate another preferred embodiment of afluorescence sensor system and a reference system comprising diffractiveoptical elements and stacked planar integrated optics; and

FIGS. 6A-6C illustrate different applications of multi-analyte sensordevices according to the invention.

DETAILED DESCRIPTION

FIG. 1 shows a flow chart diagram of information flow within amulti-analyte sensor device according to an embodiment of the presentinvention.

A light source controller 0 feeds a control signal to a light source 1producing one or more excitation signals la. Beam-adapting optics 2provide beam-adapted excitation lights 2 a,2 b to a fluorescence sensorsystem 5,6,7 and a reference system 3,6,7, respectively, saidbeam-adapting optics generally being optional. It is preferred, however,to include beam-adapting optics to more efficiently guide excitationlight to said fluorescence sensor and reference systems. A medium 4comprises analytes to be determined, e.g. O₂, CO₂, pH, and ionsdetermining salinity, in particular salt ions such as Na⁺ and Cl⁻, andtemperature. The reference system comprising reference light sources 3,e.g. a fluorophore, phosphore, or both, or one or more reflectors, whichare not influenced by the one or more analytes to any significant degreefor the measurement and the fluorescence sensor system comprisingfluorescence sensors 5 for sensing said analytes in the medium provideoptical signals 3 b and 5 a, respectively, to signal guiding optics 6.Detectors 7 detect light signals 6 a and 6 b from said light guidingoptics that originated from the fluorescence sensors 5 and the referencelight sources 3, respectively. The detectors 7 produce electrical outputsignals 7 a and 7 b corresponding to the fluorescence sensors 5 andphase reference system 3, respectively. Optionally, e.g. instead ofparallel channels, a multiplexer 8 selects sensor signals 8 a and thereference signals 8 b, respectively, for further processing. Optionally,an amplifier 9 amplifies said signals 8 a, 8 b from the multiplexer. Aphase detector 10 detects the delays between said one or more electricaloutput signals, specifically here the delay between the amplified,selected electrical output signal 9 a of the selected fluorescencesensor and the amplified electrical signal 9 b of the reference system.Here signal processor 11 further treats the output signal of the phasedetector and produces an output signal 12.

Referencing according to the present embodiment differs from that of theprior art in the phase reference system 3. According to the prior art areference signal 0 a (not part of the present embodiment according tothe invention) from the light source controller 0 is fed to the phasedetector 10 whereby drift and other contributions to the signalinformation in the various units 1,2, and 6-9 are not accounted for.

Compared with this prior art, the present invention provides a series ofreference signals 2 b, 3 b, and 6 b-9 b that are influenced by all thecontributions of drift, etc. from the units 1, 2, and 6-9.

FIG. 2 illustrates time delay Δt between an electrical sensor outputsignal 7 a and a phase reference signal 7 b in frequency domainfluorescence lifetime measurement. Further, FIG. 2 illustrates themodulation Δl of the amplitude defined as the difference in amplitude atthe time delay Δt.

Phase detection techniques are known in the art, see e.g. StanfordResearch Systems Catalogue 1998, Application Note #3, “About Lock-inAmplifiers” pp. 193-204, the content of which is hereby incorporated byreference.

FIG. 3 illustrates a preferred embodiment of a fluorescence sensor andreference system in a longitudinal side view of cross sectional alongline III-III (see FIG. 4B)

A cylindrical housing 310 b is assembled with a sensor cap comprising asensor holder 310 a and sensor lid 308 combination, here illustratedwith a screwed female-male threading fixing the housing and captogether. Other fixation techniques can be used e.g. adhesion, welding,and mechanical latching such as snap-lock latching.

Here, the exchangeable sensor cap accommodates fluorescence sensors 307a and 307 b, e.g. pO₂ (Lifetime: Ru[dpp]) and pH (Dual LifetimeReferencing (DLR): Fluorescein and Ru[dpp]), respectively and referencesystem 309, e.g. comprising a mirror as the passive reference lightsources. It can be exchanged when damaged, or reconfigured withdifferent fluorescence sensors and reference systems, when other or newapplications are required.

The housing accommodates an excitation light source, here illustrated bya light emitting diode (LED) 302 having a predominant emission at 470nm, e.g. an InGaN-based LED from Agilent (HLMP-CB15/16), and positionedin the centre of the housing, and an optional excitation filter 304 forselecting one or more wavelengths of interest, here a typical 480 nm lowpass filter is used. Suitable optical filters can be filters based oninterference, absorption, or both, or based on any other non-fluorescenttype filter.

The excitation light source is preferably adapted to transmit light in apredetermined direction, e.g. by optically shielding off unwanted lightto the optical waveguides collecting the emitted fluorescence light fromthe fluorescence sensors.

Light sources of the solid-state type are preferred for manyapplications because they might be superior to e.g. flash lamps, as theyare smaller, cheaper, have longer lifetimes, and can be modulateddirectly through a bias-current.

Generally, the excitation optics comprises traditional lenses,diffractive optical elements (DOE), fibres, diffusers, and anycombination of beam-shaping optics, alone or in combination.

The LED is modulated at 45 kHz, which is a suitable modulation frequencyfor most applications.

A light directing means, here illustrated by a reflective cone 306 ispositioned to receive excitation light at its outer surface and directit through reflection to fluorescence sensors, here illustrated byfluorescence sensors 307 a and 307 b mounted on the outer side of a caplid 310 c, and direct it to the reference system, here illustrated by amirror of reference system 309 mounted on the outer side of a cap lid308 and functioning as a passive reference light source.

Fluorescence light from the fluorescence sensors, here fluorescencesensors 307 a and 307 b positioned in the lid 308 of the exchangeablesensor cap, is guided through optical wave guides, here optical fibres303 a, 303 b, and 303 c, to detectors 300 a, 300 b, and 300 c, heresolid-state photodiodes in front of which optical filters 301 a, 301 b,301 c are placed in order to reduce stray-light induced errors.

The various components, here excitation light source 302, lightdirecting means 306, optical wave guides 303 and detectors are embeddedin a solid body thereby providing a sensing device which is rugged andinsensitive to vibrations and shock.

The solid body comprises inserts for wave-guides 311 a, 311 b, 311 c anddetectors and an insert for the excitation light source 312.

The sensor body material is chosen to be transparent in the excitationwavelength region e.g. around 470 nm. Suitable materials are known inthe art including polymers like polycarbonate and polystyrene.

Furthermore, the solid body design eliminates optics/air interfaces atwhich possible dew formations from operation in humid environments mightdisturb the signal and signal loss due to reflections are minimized.

The fluorescence light is emitted isotropically from the fluorescencesensors. It is therefore important that the collection system can detectthe emitted fluorescence light under as large an angle as possible.Here, high NA plastic fibres preferably collect the emitted fluorescencelight. Collection of the emitted fluorescence light in this way ensuresthat a high light collection efficiency is obtained whereby it possibleto excite the fluorescence sensors with light of lower intensity toobtain a comparable fluorescence light signal and thereby to diminishphoto-bleaching and prolong the lifetime of the fluorescence sensors.

FIGS. 4A-4C illustrate a preferred embodiment of an exchangeable sensorcap illustrated in FIG. 3.

FIG. 4A shows a longitudinal sectional view of the sensor cap along theline III-III (see FIG. 4B). The sensor cap comprises a cap lid 308 fixedto a sensor holder 310 a with male screw threadings. Fluorescencesensors 307 a and 307 b and reference system 309 are fixed to the outerside of the cap lid.

FIG. 4B shows a bottom view of the sensor cap comprising a sensor holder310 a and cap lid 308 to the surface of which fluorescence sensors 307 aand 307 b are fixed.

FIG. 4C shows a perspective view of the sensor cap.

Specific sensors capable of measuring pH, O₂, CO₂, salinity andtemperature by utilizing 5 different kinds of fluorescent sensorchemistries—three of them based on the Dual Lifetime Referencing (DLR)technique and 2 of them true fluorescence lifetime sensors—all availablefrom PreSens, Regensburg, Germany:

-   PO₂ (Lifetime: Ru[dpp])-   pH (DLR: Fluorescein and Ru[dpp])-   pCO₂ (DLR: HPTS and Ru[dpp])-   Salinity (DLR: Lucigenin and Ru[dpp])-   Temperature (Lifetime: Ru[phen])

For DLR and the specific fluorescence sensors and other useful sensorssee WO99/06821 the content of which is incorporated herein by reference.

FIGS. 5A and 5B illustrate another preferred embodiment of afluorescence sensor system and a reference system comprising diffractiveoptical elements and stacked planar integrated optics which areparticularly suited for cheap mass production.

The sensor is divided into multiple layers which can be produced onwafers, stacked and sliced dramatically reducing production cost andtime (see e.g. Sinzinger, S. J. J “Microoptics”, Wiley-VCH, 1999).

The sensor comprises 4 primary layers: a sensor and reference layer 53comprising fluorescence sensors and phase reference system, hereincluding a mirror, a diffractive optical element layer 52 optionallyfurther comprising one or more conventional optical elements such aslenses, diffusers, prisms, beam splitters, and coatings, a filter layer55 and a detector layer 51, and an excitation light source 54, here alight emitting diode, located above the detector layer, and anelectronic layer 50, here coupled to the detector layer 51.

The detector and filter layer can be combined in a common layer, if thefilters are deposited directly on the detector layer.

The diffractive optical element is divided into two main sections: acentre section 58 and a circumference section 59.

The centre section comprises diffractive gratings adapted to focus lightfrom excitation light source onto the different fluorescence sensors andthe phase reference system.

The circumferential section comprises a section for each fluorescencesensor and one for the phase reference system. Each section comprisesdiffractive gratings adapted for collecting light from the fluorescencesensors and phase reference system and for focussing the collected lightthrough the filter layer 55 and further on to the detector layer 51.

Diffractive optical elements are known in the art, see e.g. Babin, S. V.“Data preparation and fabrication of DOE using electron-beamlithography”, Optics and Lasers in Engineering, Vol. 29 Issue 4-5, 1998,pp. 307-324, and Taghizadeh, M. R. et al. “Design and fabrication ofdiffractive optical elements”, Microelectronic Engineering, Vol. 34,Issue 3-4, 1997, pp. 219-242.

FIG. 6 a shows a flow cell 60, where sensor chemicals of thefluorescence sensors 64 a, 64 b and reference 63 are applied to the wallthereof. The chemicals are excited through a transparent section of theflow cell through which the fluorescence light is also detected.

FIG. 6 b illustrates a micro bioreactor 61 with sensor chemicals appliedto the transparent bottom. The processes in the reactor can then bemonitored from below with one of the sensor systems previouslydescribed.

FIG. 6 c shows a micro fluid channel system 62 allowing continuouslymonitoring of very small liquid volumes.

1. An apparatus for measuring fluorescence lifetimes of fluorescencesensors for one or more analytes, the apparatus comprising (a) one ormore excitation light sources, said light sources being adapted toproduce one or more excitation signals; (b) one or more fluorescencesensor systems, said sensor systems each comprising one or morefluorescence sensors for sensing the one or more analytes and beingadapted to receive said one or more excitation signals to produce one ormore optical sensor signals in response thereto, and to produce one ormore electrical output signals in response to said optical sensorsignals, said one or more electrical output signals being delayed withrespect to said one or more excitation signals and being characteristicsof the fluorescence lifetimes of the one or more fluorescence sensors;(c) one or more reference systems, said reference systems eachcomprising one or more reference light sources and being adapted toreceive said one or more excitation signals to produce reference opticalsignals in response thereto, and to produce one or more electricalreference output signals in response to said one or more opticalreference signals; and (d) one or more phase detectors, said phasedetectors being adapted to detect one or more delays of said one or moreelectrical output signals of said one or more fluorescence sensorsystems and said one or more reference systems, and to produce one ormore phase output signals; wherein said one or more fluorescence sensorsand said one or more reference light sources are incorporated in anexchangeable sensor cap.
 2. The apparatus according to claim 1 whereinsaid one or more reference light sources comprise a fluorophore, aphosphore, or both.
 3. The apparatus according to claim 1 wherein saidone or more reference light sources comprise one or more reflectors,said reflectors reflecting said light of said one or more excitationlight sources.
 4. The apparatus according to claim 3 wherein said one ormore reflectors comprise a diffuse reflector, a retro-reflector, orboth.
 5. The apparatus according to claim 3 wherein said one or morereflectors comprise a mirror.
 6. The apparatus according to claim 1comprising a single excitation light source for said sensor systems andreference systems.
 7. The apparatus according to claim 1 comprising aplurality of excitation light sources each for respective ones of thesensor systems and reference systems.
 8. The apparatus according toclaim 1 wherein the one or more fluorescence sensors for sensing the oneor more analytes comprise a fluorophore, a phosphore, or both.
 9. Theapparatus according to claim 8 wherein the one or more analytes areselected from the group consisting of O₂, CO₂, pH, ions from an ioniccompound, and temperature.
 10. The apparatus according to claim 1,wherein said one or more fluorescence sensor systems comprise one ormore fluorescence sensors, one or more detectors, and one or more waveguides between said one or more fluorescence sensors and detectors. 11.The apparatus according to claim 1, wherein said one or morefluorescence sensor systems comprise one or more light directing means,said light directing means directing said one or more excitations lightsignals to said one or more fluorescence sensors and reference lightsource.
 12. The apparatus according to claim 11 wherein said one or morelight directing means comprise one or more reflective cones.
 13. Theapparatus according to claim 11 wherein said one or more light directingmeans comprise one or more diffractive optical elements.
 14. Theapparatus according to claim 1 wherein said one or more fluorescencesensor systems and said reference system are incorporated in a flowcell.
 15. The apparatus according to claim 1 wherein said one or morefluorescence sensor systems and said reference system are incorporatedin a micro bioreactor.
 16. The apparatus according to claim 1 whereinsaid one or more fluorescence sensor systems and said reference systemare incorporated in a micro fluid channel system.
 17. The apparatusaccording to claim 1 wherein said one or more sensors of the sensorsystems are wholly or partially covered with one or more semi-permeablemembranes.
 18. The apparatus according to claim 1 wherein said one ormore excitation light sources comprise at least one excitation lightsource adapted to operate in frequency domain.
 19. A fluorescencelifetime sensing device for sensing fluorescence light of fluorescencesensors for one or more analytes, the sensor device comprising afluorescence sensor system comprising one or more fluorescence sensorssaid sensors being adapted to sense the one or more analytes and producefluorescence light in response thereto; a phase reference systemcomprising a reference light source; an optical light beam-adaptingsystem providing excitation lights for the fluorescence sensors andreference light for said phase reference system; a detection systemcomprising detectors for detecting said fluorescence light from saidfluorescence sensors and reference light from said phase referencesystem; and an optical sensor and reference signal guiding system, saidguiding system guiding said fluorescence light, and said reference lightto said detectors; wherein said optical light beam-adapting systemcomprises a reflective surface directing said excitation light to thefluorescence sensors and said reference light to said phase referencesystem; and wherein the reflective surface is the outer surface of acone.
 20. The sensing device according to claim 19 wherein the opticallight beam-adapting system comprises optical fibres.
 21. The sensingdevice according to claim 19 wherein the optical sensor and referencesignal guiding system comprises optical fibres.
 22. A fluorescencelifetime sensing device for sensing fluorescence light of fluorescencesensors for one or more analytes, the sensor device comprising afluorescence sensor system comprising one or more fluorescence sensors,said sensors being adapted to sense the one or more analytes and producefluorescence light in response thereto; a phase reference systemcomprising a passive reference light source; an optical lightbeam-adapting system providing excitation lights for the fluorescencesensors and reference light for said phase reference system; a detectionsystem comprising detectors for detecting said fluorescence light fromsaid fluorescence sensors and reference light from said phase referencesystem; and an optical sensor and reference signal guiding system, saidguiding system guiding said fluorescence light and said reference lightto said detectors; wherein said one or more fluorescence sensors andsaid reference light source are incorporated in an exchangeable cap. 23.A fluorescence lifetime sensing device for sensing fluorescence light offluorescence sensors for one or more analytes, the sensor devicecomprising a fluorescence sensor system comprising one or morefluorescence sensors, said sensors being adapted to sense the one ormore analytes and produce fluorescence light in response thereto; aphase reference system comprising a reference light source; an opticallight beam-adapting system providing excitation lights for thefluorescence sensors and reference light for said phase referencesystem; a detection system comprising detectors for detecting saidfluorescence light from said fluorescence sensors and reference lightfrom said phase reference system; and an optical sensor and referencesignal guiding system, said guiding system guiding said fluorescencelight and said reference light to said detectors; wherein said opticallight beam-adapting system, and said optical sensor and reference signalguiding system, are incorporated in a diffractive optical element; andwherein said diffractive optical element comprises a first set ofdiffractive gratings adapted to focus light from an excitation lightsource onto respective ones of the fluorescence sensors and onto saidphase reference system; and a second set of diffractive gratings adaptedto collect light from respective ones of the fluorescence sensors andfrom the phase reference system and to focus the collected light ontosaid detectors.
 24. The sensing device according to claim 23 whereinsaid optical light beam adapting system comprises a stacked planarintegrated optical layer structure.
 25. The sensing device according toclaim 24 wherein said layer structure comprises an electronic layer, adetector layer, a light source, a diffractive optical element, a sensorand reference layer, and a filter layer.
 26. A method of measuringconcentration of one or more analytes, the method comprising (a)providing an apparatus as defined in claim 1; (b) applying said one ormore excitation light signals to said one or more fluorescence sensorsystems and to said one or more reference light sources; (c) applyingsaid one or more electrical output signals of said one or morefluorescence sensor systems and said one or more reference systems tosaid one or more phase detectors; (d) determining said one or moredelays by said one or more phase output signals; (e) comparing said oneor more determined delays with delay calibration data of knownconcentrations of the one or more analytes, thereby obtaining aconcentration value; and (f) outputting a signal representative of theconcentration value.
 27. A method of measuring concentration of one ormore analytes, the method comprising (a) providing an apparatus asdefined in claim 19; (b) applying said one or more excitation lightsignals to said one or more fluorescence sensor systems and to said oneor more reference light sources; (c) applying said one or moreelectrical output signals of said one or more fluorescence sensorsystems and said one or more reference systems to said one or more phasedetectors; (d) determining said one or more delays by said one or morephase output signals; (e) comparing said one or more determined delayswith delay calibration data of known concentrations of the one or moreanalytes, thereby obtaining a concentration value; and (f) outputting asignal representative of the concentration value.
 28. A method ofmeasuring concentration of one or more analytes, the method comprising(a) providing an apparatus as defined in claim 22; (b) applying said oneor more excitation light signals to said one or more fluorescence sensorsystems and to said one or more reference light sources; (c) applyingsaid one or more electrical output signals of said one or morefluorescence sensor systems and said one or more reference systems tosaid one or more phase detectors; (d) determining said one or moredelays by said one or more phase output signals; (e) comparing said oneor more determined delays with delay calibration data of knownconcentrations of the one or more analytes, thereby obtaining aconcentration value; and (f) outputting a signal representative of theconcentration value.
 29. A method of measuring concentration of one ormore analytes, the method comprising (a) providing an apparatus asdefined in claim 23; (b) applying said one or more excitation lightsignals to said one or more fluorescence sensor systems and to said oneor more reference light sources; (c) applying said one or moreelectrical output signals of said one or more fluorescence sensorsystems and said one or more reference systems to said one or more phasedetectors; (d) determining said one or more delays by said one or morephase output signals; (e) comparing said one or more determined delayswith delay calibration data of known concentrations of the one or moreanalytes, thereby obtaining a concentration value; and (f) outputting asignal representative of the concentration value.